The present invention relates to the field of immune adjuvants and the use thereof as immune adjuvants in vaccines. The compositions of the present invention exhibit significantly improved properties relevant to the lytic effect, tolerance to QS-21 associated pain, and product stability of QS-21, and maintain full adjuvant activity.
Adjuvant saponins have been identified and purified from an aqueous extract of the bark of the South American tree, Quillaja saponaria Molina. Among the 22 peaks which were separable and displayed saponin activity, QS-21 was one of the, more predominant purified saponins. This saponin has been substantially purified by high pressure liquid chromatography (HPLC), low pressure liquid silica chromoatography, and hydrophilic interactive chromatography (HILIC). QS-21 has been found to be useful as an immune adjuvant for enhancing immune responses in individuals at a much lower concentration than the previously available heterogeneous saponin preparations without the toxic effects associated with crude saponin preparations.
QS-21 is a membrane-lytic triterpene glycoside saponin. It forms micelles of approximately the same radius as bovine serum albumin (Kensil, U.S. Pat. No. 5,057,540) and has a critical micellar concentration of approximately 50 xcexcg/ml in PBS (Soltysik, S., et al., 1995, Vaccine 13:1403-1410).
The potency of an adjuvant formulation containing an antigen plus QS-21 can be assessed in experiments that address the relationship of adjuvant dose to immunological function (dose-response experiments). A decrease in adjuvant potency is expected to increase the minimum dose (threshold dose) required for enhancement of immune response. A desirable composition is expected to maintain an equivalent or better potency than the formulation that is used as a reference. For QS-21, the reference formulation is a simple solution in phosphate-buffered saline (PBS) or saline.
The adjuvant activity of QS-21 is assessed in animal models such as mice. The primary responses measured are increases in antigen-specific antibody and antigen-specific cytotoxic T lymphocytes (CTL). The threshold dose of QS-21 that will enhance murine immune response (antibody or CTL) has been measured in simple buffer solution such as PBS. A dose of 2.5 xcexcg has been shown to be the threshold dose for antibody (Kensil, C. R., et al., 1993, Vaccine Research 2:273-281) and for CTL (Newman, M. J., et al., 1992, J. Immunology 148:2357-2362) to the antigen ovalbumin (OVA) in C57BL/6 mice in PBS. Similar threshold doses were observed when aluminum hydroxide was included in the PBS formulation (Kensil, C. R., et al., 1993, Vaccine. Research 2:273-281). However, it is expected that there may be differences in potency between different compositions of a given adjuvant.
Despite these beneficial qualities, QS-21 possesses some unwelcome qualities as well. For instance, QS-21 associates with phospholipid bilayers and causes a lytic effect on certain cell membranes (i.e., erythrocytes). QS-21 will absorb to the phospholipid bilayer of sheep erythrocytes and cause the red blood cells to release hemoglobin. This hemoglobin release, which is known as hemolysis, occurs at a concentration of approximately 5-7 xcexcg/ml in a simple buffer such as saline or PBS (Kensil, C. R., et al., 1991, J. Immunology 146:431-437). At higher concentrations (above the critical micellar concentration of QS-21), total lysis of the red blood cell membrane occurs is, therefore, an undesirable property for a composition.
In in vivo studies, hemolysis is not noted. However, after intramuscular injection of QS-21/saline solutions into New Zealand white rabbits, mild to moderate fibroblast damage or necrosis is noted in some animals when the injection site is analyzed histopathologically (Kensil, C. R., et al., 1995, In: Vaccine Design: The Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J., Eds., Plenum Press, NY). Further, creatine kinase, a marker for muscle damage is increased after injection with QS-21 in saline or PBS. This rise is believed to be due to the lytic effect of QS-21 on cell membranes.
Moreover, in clinical trials, some individuals have experienced an immediate, transient pain after injection with QS-21 in simple buffer solutions (saline or PBS). This pain, described by most individuals as a burning pain, may be a secondary reaction correlated with the lytic effect of the QS-21 adjuvant. Patient pain is likewise an objectionable property for a composition.
Product stability is another concern for QS-21 containing compositions. The shelf life of a vaccine product is typically defined by the extent of time to reach a defined and acceptable low level of degradation (such as, the time to 10% degradation, also known as t90). Most commercial vaccine products have a shelf life of at least 18 to 24 months when stored in the refrigerator at 4xc2x0 C. Adjuvants, which are essential components of vaccines, therefore must also have equally long shelf lives. However, the shelf life of a 50 xcexcg/ml solution of QS-21 at pH 7.0 at 4xc2x0 C. is reached in about 3 months. The reason for the short shelf life is because the ester bond of QS-21 is increasingly labile at increasing pH and because monomers of QS-21, as opposed to micelles, are subject to hydrolysis. The need to stabilize compositions of QS-21 adjuvant is significant.
A need exists for compositions of the saponin adjuvant QS-21 that may be used to boost the antigenic immune response in a relatively low dose with low local reactions and side effects, but also features a reduced lytic effect, improved tolerance to QS-21, and an increased stability. Accordingly, the present invention provides novel compositions of QS-21 that have these improved characteristics compared to a simple solution of QS-21 in a buffer such as saline or PBS. Surprisingly, the full adjuvant potency of QS-21 in the disclosed compositions is not compromised compared to a control formulation of QS-21 in PBS.